Asthma is an airway disease characterized by inflammation; accumulation of mast cells, eosinophils, and neutrophils; and intermittent, reversible airway obstruction that affects more than 300 million people worldwide. The role of extracellular proteases in asthma is not well-characterized, nor are there effective, targeted therapies that exploit or block their activity. Inflammatory cells secrete proteases that are correlated with the lung pathogenesis in asthma and chronic obstructive pulmonary disease. The secreted proteases include elastase, chymase, ADAM33, and matrix metalloproteinases (MMPs) like MMP2/9/12. Evidence for their involvement in asthma primarily consists of upregulated protease mRNA or protein levels in histological sections or other postmortem tissue samples. However, this type of data does not account for the presence of complementary inhibitors, such as tissue inhibitors of metalloproteinases for MMPs. While genetic knockouts have demonstrated that a subset of these proteases, including MMP2/9, is important in asthma pathogenesis, they do not give insight into the timing and location of the protease activity. We propose to overcome these limitations with activatable cell penetrating peptides (ACPPs). ACPPs highlight in vivo proteolytic activity because their cellular uptake depends on activation by specific proteases. Preliminary data with MMP2/9- and elastase-cleavable ACPPs show that we can differentiate between asthma and control lungs and airways and that the ACPPs highlight areas of lung inflammation. Identification of the key proteases in asthma will elucidate targets for ACPP-driven therapy. By characterizing the uptake of a panel of fluorescently-labeled ACPPs in mouse models of asthma, we propose to profile the expression of proteases that may play a role in acute asthma. We predict that neutrophils and eosinophils will be labeled by ACPPs that are specific for MMP2/9 and elastase and that mast cells, airway smooth muscle cells, and fibroblasts will be labeled by ACPPs that are specific for MMP12, chymase, and ADAM33. ACPPs with fluorescent and/or non-optical (e.g., 64Cu for positron emission tomography) imaging payloads will be used for longitudinal in vivo imaging of mice at various stages of asthma initiation, progression, and maintenance to characterize the proteases that promote chronic airway remodeling. We predict that chymase and MMP2/9/12 are the drivers of chronic remodeling and will have protease activity that correlates with disease severity. We propose to increase the efficacy of asthma therapeutic agents by attaching ACPPs to glucocorticoids and protease inhibitors, such as dexamethasone and marimastat, respectively, and administering them intravenously or intranasally to mice during antigen challenge and after established disease. We will assess treatment efficacy by imaging and function testing. We hypothesize that ACPP-based delivery of these therapies will increase their therapeutic index. The application of ACPPs to mouse models of asthma will improve our understanding, evaluation, and treatment of asthma.